The present invention pertains to a composition and method for immobilizing microorganisms, catalysts, and enzymes in a gel including an anionic polysaccharide polymer and a cationic polymer, homogeneously dispersed and bonded, to form a matrix structure. In particular, the invention pertains to a method and composition in which an anionic form of alginate, polyethyleneimine, and yeast are homogeneously dispersed in an aqueous system, and thereafter mixed with an oil phase to form beads.
Recent interest has turned to increasing the productivity of bioconversion processes by utilizing "biocatalyst" systems in which cells are immobilized. In the context of the present application, the term "biocatalyst" is understood to mean a biological system including enzymes or whole cellular microorganisms fixed through a macroscopic carrier. As used herein, the term "biocatalytic entity" refers to enzymes or whole cellular microorganisms which are to be incorporated into a biocatalyst. High cell immobilization costs, limited cell service life, loss of catalytic activity and changes in biological behavior of immobilized cells have been major problems which have hindered widespread commercial use of proposed biocatalyst systems.
Currently, there are two techniques for immobilizing cells. One technique involves the use of solid supports. Typically, cells are either cultured or adhered to the surface of the solid support. Wood chips, porous brickets, Rasching rings, PVC flakes, glass fibers, porous glass, and clays have been utilized to obtain surface cultures for the production of ethanol. The use of solid supports has been limited by the problems of cell washout, limited cell loading capability, and a high rate of free cell production in the broth.
Another technique for cell immobilization has been to immobilize cells within a gel matrix usually in the form of beads or pellets. The beads or pellets present a large surface area to mediums in which the beads or pellets are suspended and allow nutrients and reaction products to diffuse into or out of a small cross-sectional area.
A common encapsulation system has utilized polyacrylamide gels. The usefulness of gels made out of polyacrylamide has been limited by the toxicity of the monomer substance and the brittleness of the gels. Hydrogels, derived from marine plants, are considered to be more desirable as a live cell carrier. Hydrogel's inertness, high water holding capacity, permeability, ease of forming and abundance are desirable features for immobilizing cells for bioconversion processes. Examples of hydrogels which have been studied extensively include alginate, carrageenan, and agar.
A typical example of forming gel beads utilizing hydrogels includes mixing an aliquot of yeast slurry in a hydrogel solution at 50.degree. C. The solution is then dripped into a cold organic solvent which reacts with the hydrogel to cause the hydrogel to gel into a bead-like structure. See: K. Toda and M. Shoda, Biotechnoloy and Bioengineering, Vol. 17, p. 481 (1975).
M. Wada, J. Kato, and I. Chibata, report in an article entitled "A New Immobilization of Microbial Cells," European Journal of Applied Microbiology, Biotechnology, Vol. 8, pp. 241-247 (1979), that beads containing yeast cells have been prepared by dripping a 4% carrageenan solution containing yeast cells into a 2% potassium chloride solution at an ambient temperature. Similarly, T. Shiotani and T. Yamane report in an article entitled "A Horizontal Packed Bed Bioreactor to Reduce CO.sub.2 Gas Holdup in the Continuous Production of Ethanol by Immobilized Yeast Cells," European Journal of Applied Microbiology, Biotechnology, Vol 1. 13(2) pp. 96-101 (1981) that yeast-alginate beads were prepared from a sodium alginate mixture dripped into calcium chloride solution. I. Veliky and R. Williams report in an article entitled "The Production of Ethanol by Saccharomyces Cervisiae Immobilized in Polycation-Stabilized Calcium Alginate Gels," Biotech. Lett., Vol. 3, pp. 275-280 (1981), the successful immobilization of microorganisms in a calcium alginate matrix which was thereafter further surface treated with polyethyleneimine (hereinafter referred to as PEI).
U.S. Pat. No. 4,355,105 to Lantero entitled "Glutaraldehyde/Polyethyleneimine Immobilization of Whole Microbial Cells" discloses that cells can be immobilized by providing an aqueous medium containing whole cells of a microorganism and adding glutaraldehyde to the aqueous medium to form a reaction product with the microorganism. Thereafter, PEI is added to the aqueous medium to flocculate the reaction product. The reaction product is recovered from the aqueous medium as a cake or pellet which can be further processed into particles or other forms.
U.S. Pat. No. 4,347,320 to Borglum entitled "Immobilization of Microorganisms in Gelled Carrageenan" discloses a method of immobilizing microorganisms by mixing the microorganisms with an aqueous solution of kappa-carrageenan. The microorganisms and kappa-carrageenan are gelled by forming droplets through a nozzle and contacting the droplets with an aqueous solution containing a gelling agent including PEI. The resultant beads have a surface skin of PEI.
Suhaila and Salleh, in an article entitled "Physical Properties of Polyethyleneimine-Alginate Gels," Biotechnology Letters, Vol. 4, No. 9, pp. 66-614 (1982) discuss the properties of PEI-propylene glycol alginate gels as compared to gelatin-propylene glycol alginate gels (hereinafter propylene glycol alginate will be referred to as PGA). The authors found that PEI-PGA gels are much more brittle than gelatin-PGA gels but maintain their integrity to a greater extent in acidic conditions and are stable to heat and freezing. While the authors suggest that PEI-PGA gels may be useful for enzyme cell immobilization within certain limitations inherent in the brittle PEI-PGA structure, there is no disclosure of an anionic polysaccharide and a cationic polymer homogeneously dispersed and bonded to form a matrix.
An article in Newswatch, p. 3, Mar. 17, 1984, entitled "Crah Chitosan Microbeads Entrap Cells in One Step, Challenge Damon 'Encapcel'," reports that Chokyun Rha of the Massachusetts Institute of Technology has produced globular spheres of 10 microns to 5 millimeters in size with controlled porosity using chitosan (a dissolved deacetylated chitin) and alginate. The article further reports that cells or enzymes are suspended in a cationic chitosan solution. Droplets of the cell-chitosan mixture are added to an anionic solution of alginate or another polymer to form membranes which encapsulate a volume of the liquid chitosan-cell mixture. The membranes can be strengthened by cross-linking with divalent ions or additional polymer layers. The capsules can withstand a 2,000-G force and undergo deformations of up to 90% without rupture.
To the best of our knowledge, these proposals have not led to biocatalytic systems utilizing beads having sufficient strength and resiliency while capable of maintaining cell viability. Materials such as glutaraldehyde and PEI have not found widespread use in biocatalytic gel matrix systems. Generally, the PEI and glutaraldehyde in solution have fungicidal and bactericidal action which greatly limits their use for incorporation into matrixes. Specifically, PEI is ionic in solution and may cause cell lysis. Accordingly, one would not predict that homogeneous incorporation of PEI within a matrix system would result in live cell systems having catalytic activity.
In the past, PEI has been used primarily to impart rigidity to beads. The PEI would be applied to the bead structure as a coating or skin such that the toxic effects of PEI would not damage the biocatalyst. Rigid or hard beads were considered better suited for packing in a column. However, rigid or hardened beads are unable to accommodate cellular growth and are incapable of venting gaseous metabolic by-products. In fermentation processes which extend over a long period of time, rigid bead structures rupture and release the biocatalyst from the gel matrix.
The prior art does not suggest a practical means for obtaining beads having a predetermined substantially uniform size. Bead size is an important consideration in biocatalytic systems. Cells incorporated within the interior of large beads cannot proliferate or contribute to fermentation processes due to the cell's inability to obtain necessary nutrients through the matrix structure. Only cells incorporated near the surface of the gel's matrix proliferate and actively contribute to fermentation processes.
Gel matrixes comprising hydrogel which utilize calcium for the formation of a cross-linking structure eventually dissolve releasing the biocatalyst. Microorganisms generally utilize the calcium within the matrix structure for their own purposes, and calcium is released from the matrix structure as part of an ongoing release upon dissolution of the bead components within an aqueous medium.